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The proposed Phase II project will continue development of gammaPNA miniprobe technology originally developed at Carnegie Mellon University and the University of Pittsburgh and subsequently transferred to PNA Innovations, Inc, a small business spun out of Carnegie Mellon University. The basis of gammaPNA miniprobes is the high affinity with which gammaPNA hybridizes to complementary DNA. The specific applications addressed in this proposal are telomere analysis, which is currently done using fluorescent PNA probes 18 bases in length, which hybridize to 3 consecutive repeats of the human telomere sequence 5'-AATGGG-3', and mRNA labeling by complementary fluorescent probes. In Phase I, we demonstrated that the higher affinity of gammaPNA allows shorter 12 base telomere probes to be used, resulting in more fluorescent dyes being delivered to a telomere of a given length. This allow more reliable analysis of the shortest (i.e. critically short) telomeres, which are implicated in a variety of conditions including aging-related diseases and cancer. We published a paper describing our results in Organic and Biomolecular Chemistry and we launched a marketing campaign around our Telo MiniprobesTM, leading to our first sales within this product line. The proposed research will have four Specific Aims.
The first aim extends our Phase I work in three ways. First we will study several additional cell lines This will help us to (a) determine the range of variability in miniprobe performance and (b) potentially identify other versions of the miniprobe that work in varied cell lines. Second, we wil develop miniprobes that target the C-rich telomere strand. Third, we will build on promising preliminary results for synthesizing internally labeled miniprobes that will double or triple the brightness of our current best probe. The second and third aims are directed toward new applications, specifically in development of a high throughput telomere assay based on our miniprobes and testing of fresh and archived tissue samples, which are currently difficult to study by FISH due to autofluorescence.
The fourth aim will significantly extend gammaPNA FISH probes into RNA labeling. We will synthesize sets of gammaPNAs targeted to different sites on a single mRNA, but rather than covalently label the probes, which is costly, we will use an innovative and economical co-hybridization approach to label our probes. The synthesis of the gammaPNA monomers and oligomers will be done at PNA Innovations. Biophysical characterization and telomere staining will be done at academic laboratories at Carnegie Mellon and the University of Pittsburgh where the gammaPNA miniprobe technology was invented. Optimized miniprobes will then be sent to independent beta-testing laboratories that currently use conventional PNA probes for telomere analysis or DNA-based molecular beacons for mRNA labeling.

Public Health Relevance

The goals of this project are to further develop a new class of fluorescent in situ hybridization (FISH) probes for telomere analysis and to extend this technology to RNA FISH applications. The technology is based on work done in two academic laboratories and is well suited to commercial development, as is the goal of PA-15-052, 'Lab to Marketplace: Tools for Biomedical and Behavioral Research'. The probes will be based on gammaPNA, an analogue of peptide nucleic acid that exhibits superior affinity and solubility. The higher affinity of gammaPNA allows shorter 'miniprobes' to be used. The ability to hybridize shorter probes means that more probes can hybridize to a given telomere, resulting in brighter fluorescence. This significantly improves the analysis of critically short telomeres, which are correlated with a variety of aging-related diseases, cancer and other conditions. New applications in this proposal include (1) development of a high-throughput telomere analysis assay, (2) testing of miniprobes for telomere analysis in fresh and archived tissue samples, and (3) generation of sets of probes for labeling non-repetitive targets such as mRNAs. The miniprobes will be synthesized at PNA Innovations Inc, then tested at Carnegie Mellon University and the University of Pittsburgh. Optimized miniprobes will then be delivered to beta testing laboratories for independent testing and feedback.